Shaped illumination geometry and intensity using a diffractive optical element

ABSTRACT

A method and apparatus to illuminate a target. The apparatus can comprise a first lens configured to receive light from the light source, a diffractive optical element configured to receive the light from the first lens and to regulate the light into regulated light, and second lens configured to receive the regulated light and to direct the regulated light onto a selected area of the target.

This is a continuation of application Ser. No. 09/964,778, filed Sep.28, 2001 now U.S. Pat. No. 6,744,502, which is incorporated herein byreference.

I. FIELD OF THE INVENTION

This invention relates to methods and optical systems for illuminating atarget. The present invention also relates to methods and systems forperforming sample assays, and for producing and measuring opticalresponses and signatures.

II. BACKGROUND OF THE INVENTION

Targets, such as areas where optically transduced chemical and/orbiochemical assays are performed, may need to be illuminated by a lightsource. It is often desirable to illuminate the target with light havingenhanced uniformity of intensity over the entire target region. Opticalsignals are typically a function of the illumination intensity and, themore an illumination intensity varies across a target, the more theoptical signal will also vary. The resultant variance in optical signalsmay be undesirable.

However, it can be difficult to efficiently provide illumination havingenhanced or a high degree of uniformity. For example, lasers, which arecommonly used for illuminating targets, typically have an intensityprofile that is peaked at its center and which drops off radiallytowards the edges. This intensity profile is often a Gaussian, or bellshaped, profile. Therefore, if a target is directly illuminated withsuch a laser, the illumination of the target will not have a constantintensity. Rather, the center portion of the target will receive greaterillumination intensity than the perimeter areas.

Therefore, there is a need for an apparatus and method to illuminate atarget or selected area with enhanced uniformity as compared to directlyilluminating the target with a given light source. Further, there is aneed for an apparatus and method that provides enhanced illuminationuniformity for optical targets such as those in chemical and/orbiochemical assay systems.

III. SUMMARY OF THE INVENTION

According to certain embodiments of the invention, an apparatus isprovided to illuminate a target. The apparatus comprises a light source,a first lens, a diffractive optical element, and a second lens. Thefirst lens is configured to receive light from the light source. Thediffractive optical element is configured to receive the light from thefirst lens and to regulate the light into regulated light. The secondlens is configured to receive the regulated light and to direct theregulated light onto a selected area of the target.

According to certain embodiments of the invention, a method is providedto illuminate a target. The method comprises generating light from alight source, directing the light with a first lens to a diffractiveoptical element, generating regulated light with the diffractive opticalelement, and focusing the regulated light with a second lens onto aselected area of the target.

According to yet another aspect of the present invention, the inventiveapparatus and method provide non-normal angle of incidence illuminationof a selected area with a given light source with a greater degree ofuniformity than is achieved when that light source is used to directlyilluminate the selected area at the same non-normal angle of incidence.

According to certain embodiments of the present invention, the inventiveapparatus and method are directed towards the analysis of a sample inwhich light is generated from a light source, the light is directed witha first lens to a diffractive optical element; regulated light isgenerated with the diffractive optical element; the regulated light isdelivered onto a selected area of a target that comprises at least oneoptically active species; and changes in an optical signature of the atleast one optically active species are detected.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention.

FIG. 1 is a schematic illustration of one embodiment of an apparatus ofthe present invention.

FIG. 2 is a schematic illustration of the illumination of a selectedarea at a non-normal angle of incidence, where the regulated light has agradient intensity profile in order to provide substantially uniformillumination of the selected area at a non-normal angle of incidence.

FIGS. 3A and B illustrate the distortive effect of non-normal angleillumination of a selected area and the use of regulated light tocompensate for this effect in order to more uniformly illuminate aselected area of a given shape.

FIG. 4 is a schematic illustration of an embodiment according to thepresent invention, having an optical diffuser for removing specklearranged between the first lens and the diffractive optical element.

V. DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

The section headings used herein are for organizational purposes only,and are not to be construed as limiting the subject matter described.All documents cited in this application, including, but not limited topatents, patent applications, articles, books, and treatises, areexpressly incorporated by reference in their entirety for any purpose.

It should be understood that the phrases “uniform illumination” and“uniformly illuminate,” as used herein with respect to the illuminationof a selected region, refer to the variation in optical intensity of thelight across the selected region. The lower the variation, the moreuniform the illumination. Thus, uniform illumination can becharacterized qualitatively as a relative measurement. The variation ofthe illumination can also be characterized quantitatively, for example,by the relative deviation of the optical intensity (“intensityvariation”) across the selected region, with smaller intensityvariations meaning that the illumination has a higher degree ofuniformity.

The relative deviation of the intensity can be found from the ratio ofthe standard deviation of the intensity to the mean value of theintensity, each measured within the selected region. The relativedeviation may also characterize a scaled intensity variation, which iscalculated from the ratio of the standard deviation of a scaledintensity to the mean value of the scaled intensity, each measuredwithin the selected region. The scaling factor may be, for example, therelative depth of each point in the selected region. The selected regionmay be selected target(s), selected area(s), and any selectedlocation(s) or device(s), and may be a single continuous area ormultiple spatially separated areas.

According to certain embodiments, the relative deviation can be measuredby dividing the selected area into a plurality of sub-areas or points,measuring a local optical intensity at each sub-area or point,determining from the plurality of local measurements a mean localoptical intensity and a standard deviation of the mean, and determiningthe relative deviation as a ratio of the standard deviation to the mean.According to certain embodiments, the relative deviation may also beexpressed in terms of percent relative deviation, by determining theproduct of 100 and the ratio of the standard deviation to the mean.

According to certain embodiments, the relative deviation can be measuredusing, for example, a light detection device, such as a two-dimensional,multi-element photodetector, such as a Charge Coupled Device (CCD). Incertain embodiments, the local optical intensity can be measured at someor all of the multiple elements of the photodetector, and the relativedeviation can be determined thereform.

The phrases “substantially uniform illumination” and “substantiallyuniformly illuminate,” as used herein, mean that the uniformity of theillumination of the selected region is greater (and its intensityvariation is less) than the uniformity of the light as initially emittedfrom the light source. That is, for a given light source, theillumination uniformity in the selected region using substantiallyuniform illumination is greater than the illumination uniformity thatresults from the direct illumination of the selected region with thegiven light source. For example, if the light source is a laser thatemits light with a Gaussian intensity distribution, the uniformity ofthe substantially uniform illumination at the selected region is greaterthan a Gaussian distribution. Likewise, if the light source has a radialintensity distribution of 1/r, the uniformity of the substantiallyuniform illumination at the selected region is greater than a 1/rdistribution.

The light intensity uniformity for “substantially uniform illumination”and “substantially uniformly illuminate” may also be scaled by theshape, including the depth, of the selected region. Thus, for example,if the selected region includes one or more wells with a curved depthprofile, the uniformity of the substantially uniform illumination can bematched to the curved profile and, for example, provide more intenseillumination where the well is deeper and less intense illuminationwhere the well is shallower. In this case, if the intensity distributionis scaled by, for example, the variable depth of the selected region,the scaled intensity distribution will be substantially uniform, meaningthat it is at least greater than the uniformity of the light intensityas initially emitted from the light source when similarly scaled.

The terms “intensity profile” and “light intensity distribution” as usedherein in reference to light, refer to the distribution of opticalintensity in the cross-sectional area of the light, where thecross-section is perpendicular to the light's propagation axis. As isthe case with illumination, the intensity profile may have a givendegree of uniformity, as measured, for example, by the relativedeviation of the optical intensity (i.e., intensity variation) in thecross-sectional area. An intensity profile may have any number ofcharacteristics, including, but not limited to, shape, total intensity,maximum intensity, minimum intensity, mean intensity, and intensityvariation. An intensity profile thus may have any cross-sectional shapeand any distribution of optical intensity in the cross-sectional area.For example, an intensity profile may be substantially uniform, that is,having essentially no measurable intensity variation in thecross-sectional area of the light. Additionally, an intensity profilemay have a gradient intensity profile, in which case the opticalintensity in the cross-sectional area will contain a range ofintensities, where the range of intensities changes in a gradient orgradated pattern from one level of intensity to another level ofintensity.

“Illumination efficiency,” as used herein, refers to the opticalintensity incident on the selected area relative to light emitted by thelight source. In certain embodiments, illumination efficiency can bemeasured, for example, by measuring with a photodetector the intensityof light incident on the selected area and the intensity of lightemitted from the light source, and taking the ratio of these twomeasurements. According to certain embodiments, the illuminationefficiency may be expressed in terms of percent relative illuminationefficiency, by determining the product of 100 and the ratio of theoptical intensity incident on the selected area relative to opticalintensity emitted from the light source.

“Numerical aperture,” as used herein, refers to a measure specifying theresolving power of an optical lens system, calculated by multiplying therefractive index of the medium occupying the lens system space by thesine of the angle between the most oblique ray entering the lens systemand the optical axis. “F-number,” as used herein, is the expressiondenoting the ratio of the equivalent focal length of a lens to thediameter of its entrance pupil, and is equivalent to 1/(2 NA), where NAis the numerical aperture. “Depth of field,” as used herein, refers tothe distance range over which light can be focused on a given subjectwhile providing adequate definition and/or clarity.

The term “edge sharpness,” as used herein, refers to the change inintensity at the edge of the region selected for illumination. Accordingto certain embodiments of the present invention, the edge sharpnessoptionally may be selected to be greater than the edge sharpness of thelight as emitted from the light source. For example, if the light sourceis a laser source emitting light with a Gaussian intensity distribution,the edge sharpness of the regulated light optionally may be selected tobe greater (that is, change more quickly at the edge of the selectedregion) than the originally emitted light. Additionally, the regulatedlight may have, for example, an edge sharpness that is approximatelysquare shaped. The regulated light may have, for example, an edgesharpness that is matched to the shape, including the depth, of theselected region. Thus, for example, if the selected region includes oneor more wells with curved depth profiles, the edge sharpness optionallycan be matched to the curved profile of the one or more wells in orderto provide more intense illumination where the well is deeper and lessintense illumination where the well is shallower. Of course, the inverseintensity distribution (that is, more intense where the well is shallowand less intense where it is deep) is also possible.

The term “optically active species,” as used herein, refers not only tospecies that rotate the plane of vibration of plane-polarized light(e.g. birefringent species), but also to species that interact withlight and which at least one of absorb light (e.g., dyes) and emit light(e.g. fluorophores or other luminescent species, quantum dots, andcolloidal particles). As used herein, it is understood that “light,”“optical,” and grammatical variants thereof are not limited to visibleradiation. For example, “light” and “optical” include, but are notlimited to, ultraviolet (UV), visible, and infrared (IR) radiation.

“Luminescence” and grammatical variations thereof, as used herein,refers to the process of absorbing light followed by subsequentlyemitting light at a different wavelength. Luminescence thus includesboth fluorescence and phosphorescence, as well as both single andmulti-photon processes.

“Optical signature,” as used herein, refers to the specific interactionsof an optically active species with light. For example, if the opticallyactive species absorbs light, the absorption spectrum of the specieswould be a component of its optical signature. Additionally, if theoptically active species emits light, the emission spectrum of thespecies would be a component of its optical signature. The opticalsignature may, of course, have any number of components. Measurement ofan optical signature, however, may include only the measurement of asingle component or subcomponent of the optical signature.

The optical signature of an optically active species may change. Forexample, it may change in response to changes in its environment, itsinteraction with another optically active species, and/or its responseto optical excitation. A change in optical signature can occur due to anumber of different mechanisms, including, but not limited to, thebinding of a dye-tagged analyte to the optically active species orsubstrate carrying the optically active species, the production of a dyespecies on or near the optically active species, the destruction of anexisting dye species, a change in optical signal upon analyteinteraction with a dye on the optically active species or substratecarrying the optically active species, or any other opticalinterrogatable event (i.e., anything that can be measured or probed withlight). Changes in an optical signature are referred to herein as an“optical response,” which is understood to further include any and allinteractions of the optically active species with light (e.g.,absorption, luminescence, birefringence). Measurement of an opticalresponse, however, may include only the measurement of a singlecomponent or sub-component of the optical response, or a single changein the optical response.

An optically active species may comprise an “indicator molecule,” whichis understood to be any molecule which can be used to determine thepresence of amplification product during or after an amplificationreaction. The skilled artisan will appreciate that many indicatormolecules may be used in the present invention. For example, accordingto certain embodiments, indicator molecules include, but are not limitedto, fluorophores, radioisotopes, chromogens, enzymes, antigens, heavymetals, dyes, magnetic probes, phosphorescence groups, chemiluminescentgroups, and electrochemical detection moieties.

A “fluorescent indicator” is any molecule or group of molecules designedto indicate the amount of amplification product by a fluorescent signal.In certain embodiments, such fluorescent indicators are “nucleic acidbinding molecules” that bind or interact, e.g., through ionic bonds,hydrophobic interactions, or covalent interactions with nucleic acid.Complex formation with the minor groove of double stranded DNA, nucleicacid hybridization, and intercalation are all non-limiting examples ofnucleic acid binding for the purposes of this invention. In certainembodiments, such fluorescent indicators are molecules that interactwith double stranded nucleic acid. In certain embodiments, fluorescentindicators may be “intercalating fluorescent dyes,” which are moleculeswhich exhibit enhanced fluorescence when they intercalate with doublestranded nucleic acid. In certain embodiments, “minor groove bindingfluorescent dyes” may bind to the minor groove of double stranded DNA.In certain embodiments, fluorescent dyes and other fluorescent moleculescan be excited to fluoresce by specific wavelengths of light, and thenfluoresce in another wavelength. According to certain embodiments, dyesmay include, but are not limited to, acridine orange; ethidium bromide;thiazole orange; pico green; chromomycin A3; SYBR® Green I (see U.S.Pat. No. 5,436,134); quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YOPRO®); and quinolinium,4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (TOPRO®). SYBR® Green I, YOPRO®, and TOPRO® are available fromMolecular Probes, Inc., Eugene, Oreg.

According to certain embodiments, the present invention provides anapparatus configured to illuminate a target. The apparatus comprises alight source, a first lens, a diffractive optical element, and a secondlens. The first lens is configured to receive light from the lightsource. The diffractive optical element is configured to receive thelight from the first lens and to regulate the light into regulatedlight. The second lens is configured to receive the regulated light andto direct the regulated light onto a selected area of the target.

According to certain embodiments, the present invention provides amethod to illuminate a target. This method comprises generating lightfrom a light source, directing the light with a first lens to adiffractive optical element, generating regulated light with thediffractive optical element, and focusing the regulated light with asecond lens onto a selected area of the target.

FIG. 1 schematically illustrates an apparatus according to certainembodiments of the invention. Light source 10 emits light 20, which isreceived by the first lens 30. The light 20 is directed by first lens 30to the diffractive optical element 40. In these illustrativeembodiments, the light 20 emitted from the light source 10 is divergentwith an angle of divergence α, and has an intensity profile that isbrightest in the center, as shown by the shading profile, which isdarkest at the center. In these illustrative embodiments, the first lens30 is also configured to collimate the light 20, as shown schematically.

The diffractive optical element 40 receives the light 20 and regulatesit into regulated light 50. In this illustrative embodiment, theregulated light 50 has an intensity profile that is substantiallyuniform, as shown by the substantially uniform shading profile. In theseembodiments, the diffractive optical element 40 is also configured toreshape the light 20 from an elliptical shape into rectangular shapedregulated light 50. The second lens 60 receives the regulated light 50and directs it onto a selected area 75 of the target 70. In thisillustrative embodiment, the second lens 60 is configured to focus theregulated light 50 towards the selected area 75, as shown schematically.

In certain embodiments, the type and properties of the light source,first lens, diffractive optical element, and second lens may be chosentogether, such that the desired shape, size, and/or uniformity isachieved for illumination of the target. Additional considerations foreach of these elements will also be discussed below.

The light source may be any effective light source that, functionally,emits light. In certain embodiments, characteristics of the light sourcemay include one or more characteristic chosen from, for example, a fixedor tunable output wavelength or wavelength range; a fixed or tunableoutput intensity; a stable, oscillating or pulsed output; a collimatedor un-collimated output; a fixed or variable divergence; and a coherentor incoherent output.

According to certain embodiments, the light source may be wholly orpartially coherent or incoherent, and may be chosen from, for example,at least one of the following non-limiting examples: a laser, anelectroluminescent light source, a chemoluminescent light source, anelectrochemoluminescent light source, an incandescent light source, afluorescent light source, an arc lamp, and a light emitting diode.According to certain embodiments, the light source may also be chosenfrom continuous wave (CW) and pulsed lasers and from gas, solid state,fiber optical, and organic based lasers.

According to certain embodiments, the light source may comprise one ormore light sources. For example, the light source may comprise a firstlight source and a second light source. In embodiments comprising morethan one light source, the light sources may be independently chosenfrom any effective light sources. Additionally, in embodimentscomprising more than one light source, such as first and second lightsources, the first source may emit light having a first opticalspectrum, which is different from a second optical spectrum of lightemitted from the second light source. As used herein, optical spectruminclude any spectrum having radiation wavelengths anywhere in at leastone of the ultraviolet, visible, and infrared regions of theelectromagnetic spectrum. According to certain embodiments, multiplelight sources, including multiple light sources having different opticalspectra, can be used, for example, to illuminate different opticallyactive species having different absorption spectra and/or to performwavelength based spectroscopic analysis. According to certainembodiments, multiple light sources, including multiple light sourceshaving different optical spectra, can be used, for example, in asimultaneous or sequential manner.

According to certain examples, selection of the light source may bebased, in whole or in part, on, for example, one or more of theproperties of the light source, including a fixed or tunable outputwavelength or wavelength range; a fixed or tunable output intensity; astable, oscillating or pulsed output, a collimated or un-collimatedoutput; fixed or variable divergence; and a coherent or incoherentoutput.

According to certain embodiments, the selection of the light source mayalso be based, in whole or in part, on one or more of the properties ofthe diffractive optical element, including its efficiency, its effectivewavelength value or range, and its angular acceptance angle. Accordingto certain embodiments, for example, the divergence of the light sourcemay be matched to the angular acceptance of the diffractive opticalelement. According to certain embodiments, the selection of the lightsource may be based, in whole or in part, on one or more of theproperties of the sample and/or the optically active species, includingtheir optical properties, such as their absorption and scatteringspectra. According to certain embodiments, the selection of the lightsource may be based, in whole or in part, on the combined properties ofthe optical system, including, for example, the collimating and/ordiverging properties of the first lens in combination with the angularacceptance of the diffractive optical element.

According to certain embodiments of the present invention, the firstlens may be configured to collimate the light from the light source, asshown schematically in FIG. 1. However, according to certainembodiments, the first lens may be configured to diverge or focus thelight from the light source. According to certain embodiments, the firstlens may be a lens system, which may be configured, for example, tocollimate the light from the light source and/or to adjust thedivergence of the light from the light source to match the angularacceptance of the diffractive optical element.

According to certain embodiments, the second lens may be configured tofocus the regulated light to substantially match a size of the selectedarea, as shown schematically in FIG. 1. However, according to certainembodiments, the second lens may be configured to collimate or divergethe regulated light. According to certain embodiments, the second lensmay be a lens system, which may be configured to collimate the regulatedlight and to reduce the regulated light to a size substantially matchedto a size of the selected area. According to certain embodiments, thesecond lens may also comprise an objective lens, which may or may not befurther configured to collect light from the selected area. According tocertain embodiments, an objective lens may be used, for example, foreither one or both of the non-normal axis illumination of the selectedarea and the collection of light (e.g., luminescent emission) from theselected area.

The first and second lenses of the apparatus may be independently chosenfrom any suitable optical element or combination of elements.Functionally, a lens bends light rays causing them to, for example, atleast one of converge and diverge. A lens may be an object or group ofobjects, and may be at least one of reflective, refractive, anddiffractive. According to certain embodiments, the first and secondlenses may be independently selected from the following non-limitingexamples: refractive optical elements, reflective optical elements, anddiffractive optical elements. According to certain embodiments, thefirst and/or second lens may be combined integrally, such as on asurface, or non-integrally with one or more other optical elements, suchas a diffractive optical element; a grating, such as a transmissiongrating; an optical filter; and a refractive element, such as a prism.According to certain embodiments, the first and/or second lens may becombined integrally, such as on a surface, or non-integrally with one ormore other non-optical elements, such as a sample holder, a fluidicsystem, a mounting system, and/or a target. According to certainembodiments, the first and/or the second lens may be disposable. Forexample, the second lens may be integrally formed in a disposable sampleholder and/or target, such as a sample holder and/or target made by, forexample, injection molding.

According to certain embodiments, the first lens and the second lens maybe chosen from one or more cylindrical lenses. According to certainembodiments, the cylindrical lens or lenses, may be used, for example,to selectively or preferentially adjust the divergence of light alongone axis of the light. According to certain embodiments, at least onecylindrical lens may be used, for example, with a light source thatemits elliptically shaped light.

According to certain embodiments, selection of the first and second lensand their properties may depend on one or more of the followingnon-limiting criteria: the properties of the light source, such as itswavelength and intensity; the diffractive optical element, including itsdiffractive properties; the sizes and shape, including divergence, ofthe regulated light; the size, shape, distance, and angle of the target;a depth of focus; a numerical aperture; an optically active species; abandwidth of the light source; and the optical path lengths and angleswithin the apparatus, including the target distance.

Diffraction optical elements are optical elements that use diffraction(the bending of light as it passes an obstruction) to controlwavefronts. Diffractive optical elements include but are not limited-todiffraction gratings, surface-relief diffractive lenses, holographicoptical elements and computer-generated holograms. Diffractive opticalelements can be formed using, for example, at least one of diamondmachining, interference of coherent beams (holography), injectionmolding, fixed and adjustable spatial light modulators, including, forexample, liquid crystal spatial light modulators, and advancedmicrolithographic techniques.

The term diffractive diffuser, and grammatical variations thereof, asused herein is understood to mean an optical element that comprises atleast one diffractive optical element but does not consist solely of, asthe at least one diffractive optical element, a diffraction grating. Incertain embodiments, a diffractive diffuser may diffract light in morethan one dimension, such as, for example, two dimensions and threedimensions.

The term diffractive optical element, as used herein, encompasses bothdiffractive diffusers and diffractive optical elements that consistsolely of a diffraction grating.

According to certain embodiments of the present invention, thediffractive optical element is configured to receive light of a givenshape and intensity distribution, and to redistribute the light toproduce a desired shape and/or intensity distribution. In certainembodiments, the redistribution of the light can be based on opticaldiffraction alone. In certain embodiments, the redistribution of thelight can be based on optical diffraction in combination with otheroptical processes, such as optical reflection and/or refraction. Opticalrefraction is a change in the direction of propagation when a wavepasses from one medium to another of different density or refractiveindex.

According to certain embodiments of the present invention, thediffractive optical element is configured to regulate the light receivedfrom the first lens. According to certain embodiments, functionally, thediffractive optical element is configured to receive light of a givenshape and intensity distribution, and to redistribute the light toproduce a desired shape and/or intensity distribution. Theredistribution results from diffraction of the light by the diffractionoptical element. The meaning of regulated light, as used herein, islight that has been redistributed from a first cross sectional shapeand/or intensity distribution to a second cross sectional shape and/orintensity distribution.

According to certain embodiments, the redistribution may includewavelength dependent redistribution. In certain embodiments, forexample, the relative wavelength distribution of the regulated light ata first point in its cross section may differ from at least one of (1)the relative wavelength distribution at a second point in its crosssection and (2) an average wavelength distribution of the light receivedby the diffractive optical element.

According to certain embodiments, the redistribution may include no (orminimal) wavelength dependent redistribution. In certain embodiments,for example, the relative wavelength distribution of the regulated lightat a first point in its cross section does not differ (or differsignificantly for the purpose of illuminating the target) from at leastone of (1) the relative wavelength distribution at a second point in itscross section and (2) an average wavelength distribution of the lightreceived by the diffractive optical element.

According to certain embodiments, diffractive optical elements can givea desired intensity distribution in the diffraction plane. According tocertain embodiments, these elements can be designed using a number ofmethods, including geometrical optics methods anditerative-Fourier-transform algorithms (IFTAs). IFTAs can be used todesign diffractive optical elements producing any desired intensitydistribution in the diffraction plane based on any intensitycross-section of the incident beam. See, for example, M. Johansson etal., “Robust design method for highly efficient beam-shaping diffractiveoptical elements using an iterative-Fourier-transform algorithm withsoft operations,” Journal of Modern Optics, 47(8), 1385–1398 (2000), theentirety of which is incorporated herein by reference for any purpose.According to certain embodiments, optical elements, and theircombinations, including, for example, diffractive optical elements, canbe designed using software packages, such as, for example, ZEMAX® fromFocus Software, Inc.

Diffractive optical elements may include, e.g., holograms andholographic optical elements. According to certain embodiments, suitablediffractive optical elements include, for example, those chosen fromamplitude (e.g., absorption) and phase holograms; optically etcheddiffractive optical elements; embossed diffractive optical elements;molded diffractive optical elements; chemically etched diffractiveoptical elements; thin or surface (2-dimensional) holographic opticalelements and volume (3-dimensional) holographic optical elements;reflection and transmission holograms; multiplex holograms; rotatingholograms, such as, for example, a rotating disc composed of a series ofholographic optical elements that diffracts light at various angles,when spinning, for example, to generate a raster scan; Fresnelholograms; and combinations thereof.

According to certain embodiments, the diffractive optical element isconfigured to regulate the received light and compensate for at leastone of light intensity distributions and shapes of the light due to atleast one of the light source and interaction of the light with opticalelements of the apparatus. As a non-limiting example, the diffractiveoptical element can be configured to redistribute the Gaussian intensityprofile of light from a laser to another intensity profile, such as amore uniform intensity profile. As another non-limiting example, thediffractive optical element can be configured to redistribute thecircular profile of light from a light source to another shape, such asa rectangular profile. As yet another non-limiting example, thediffractive optical element can be configured to compensate foraberrations, such as spherical and chromatic aberrations, such as thosecaused by the interaction of the light with optical elements of theapparatus.

According to certain embodiments, the diffractive optical elementregulates the received light, having a given degree of uniformity, suchthat the output light has a greater degree of uniformity at the selectedarea of the target. According to certain embodiments, the enhanceduniformity may be measured with respect to the intensity distribution.That is, the output light may have a more uniform intensity distribution(measured at the selected area) than the received light (if the receivedlight were transmitted to and measured at the selected area withoutbeing regulated by the diffractive optical element).

According to certain embodiments, the diffractive optical element mayregulate the received light by reshaping the cross sectional profile ofthe light. For example, the diffractive optical element may reshape thereceived light from a generally circular or elliptical cross section toform regulated light having a generally rectangular cross section. Ofcourse, the diffractive optical element may be configured to regulatereceived light having shapes other than circular, and to generateregulated light having shapes other than rectangular.

For example, as shown in FIG. 1, light 20 having an ellipticalcross-section and an intensity distribution peaked in the center (asshown by darkened central shading) is regulated by the diffractiveoptical element 40 and regulated into regulated light 50 that has arectangular cross-section and a constant intensity distribution (asshown by uniform shading). Of course, as discussed above, according tocertain embodiments the light need not be regulated into regulated lighthaving a constant intensity distribution.

The resultant intensity re-distributed and/or re-shaped light may or maynot be immediately suitable for the illumination of the selected area ofthe target. For example, in certain embodiments, the diffractive opticalelement may be configured to not only post-compensate for effects ofelements optically prior to the diffractive optical element but also topre-compensate for effects of elements optically subsequent to thediffractive optical element. Thus, according to certain embodiments, theregulated light is most suitable for illumination of the selected areaafter it interacts with the second lens and/or other subsequentelements. Additionally, according to certain embodiments, the regulatedlight may be designed to allow for its propagation, including anychanges in size, shape, and intensity distributions that resulttherefrom, prior to its illumination of the selected area.

According to certain embodiments, the diffractive optical element may beconfigured to produce regulated light that is less uniform than thereceived light but which, after interacting with subsequent opticalelements and/or further propagation, is more uniform when it illuminatesthe sample. According to certain embodiments, the diffractive opticalelement may be configured to produce regulated light having a crosssection that is not matched (in, for example, size and/or shape) to theselected area of the target but which, after interacting with subsequentoptical elements and/or further propagation, is matched (in, forexample, size and/or shape) to the selected area.

Certain embodiments of the apparatus may be configured to substantiallyuniformly illuminate the selected area without the regulated lightinteracting with a second lens. According to certain embodiments, suchapparatuses may be advantageous to illuminate selected areas atdistances that are large compared with the beam diameter. According tocertain embodiments, such apparatuses may be advantageous to illuminateselected areas at distances that are at least 1.5 times as large as thebeam diameter. According to certain embodiments, such apparatuses may beadvantageous to illuminate selected areas at distances that are at least2 times as large as the beam diameter. According to certain embodiments,such apparatuses may be advantageous to illuminate selected areas atdistances that are at least 10 times as large as the beam diameter.According to certain embodiments, such apparatuses may be advantageousto illuminate targets comprising multiple, spatially separate selectedareas.

Suitable diffractive optical elements include, but are not limited to,reconfigurable holographic optics, such as those disclosed in U.S. Pat.No. 6,175,431 to Waldern et al., which is incorporated by referenceherein in its entirety for any purpose. Diffractive optical elementsaccording to certain embodiments of the invention may be designed andselected for the specific application, and may be prepared according tomethods including, but not limited to, those disclosed in U.S. Pat. No.6,163,390 to Kanda et al., which is incorporated by reference herein inits entirety for any purpose. Diffractive optical elements for use inthe present invention may also be produced by the methods disclosed inU.S. Pat. No. 6,151,143 to Hart et al., and U.S. Pat. No. 6,111,670 toHattori et al., which are both incorporated by reference herein in theirentirety for any purpose.

According to certain embodiments, light can be shaped into virtually anyshape. According to certain embodiments, the regulated light may beshaped to match a size and shape of the selected area. Such embodimentstypically provide efficient illumination of the selected area, sincelight is not wasted by illuminating an area larger than the selectedarea. According to certain embodiments, the regulated light may beshaped such that it will match a size of the selected target area afterdiverging or converging towards the target, such as, for example, afterfocusing of the regulated light by the second lens.

According to various embodiments, a selected area can be illuminated atvirtually any angle of incidence. Thus, according to certainembodiments, the selected area may be illuminated at normal incidence(that is, at about 90° with respect to a surface plane of the selectedarea). According to certain embodiments, a selected area can beilluminated at a non-normal (“tilted” or “off-axis”) angle of incidence.According to certain embodiments, a selected area, such as a selectedarea with a non-flat surface, can be illuminated at a range of angles,including normal and non-normal angles of incidence. Further, accordingto certain embodiments, the regulated light may be designed tocompensate for the illumination of the selected area at a non-normalangle of incidence.

To achieve substantially uniform illumination of a target, such as atilted target, in certain embodiments, the diffractive optical elementis configured to provide regulated light that has an intensity gradient.For example, according to certain embodiments, as shown by shading inFIG. 2, when the regulated light is converging towards the target at anon-normal angle of incidence, the intensity profile of the regulatedlight 50 is greater towards one edge (as shown by the darker shading,which indicates greater light intensity) in order to providesubstantially uniform illumination intensity across the selected area 75of the target 70, which is illuminated at an angle tilted from normal byangle β. In certain embodiments, the intensity gradient may beproportional to the tilt angle, with the graded intensity increasingtowards the edge furthest away from the selected area.

According to certain embodiments, the apparatus may be configured toilluminate a selected area of a given shape at a normal or non-normalangle of incidence. For example, in certain embodiments, there can benon-normal illumination. In certain embodiments, if the selected areais, for example, square shaped, the regulated light would be shaped suchthat, when incident on the selected area at the non-normal angle ofincidence, the light illuminates a square shaped area. Due to thenon-normal angle of incidence, however, the regulated light will notnecessarily have a square shaped profile.

According to certain embodiments, the non-normal angle illumination of aselected area with light of a given cross sectional profile hasanalogies with the shape distorted shadow of an object cast on to asurface at a non-normal angle. For example, as shown in FIG. 3A, asquare object 110 can cast, at a non-normal angle, a shadow 115 that isa skewed trapezoid. Also, a trapezoidal object 120 can cast, at anon-normal angle, a shadow 125 that is a parallelogram. Thus, if acertain shaped shadow is desired (e.g., parallelogram), then the shapeof the object can be selected or designed to produce this shadow whencast at a specific angle.

Similarly, in certain embodiments, the present invention may illuminatea rectangular shaped selected area of the target at a non-normal angleof incidence. According to certain embodiments, the diffractive opticalelement may be configured to generate trapezoidal shaped regulated lighthaving an optical intensity gradient increasing toward a shorterparallel side of the trapezoidal shaped regulated light. For example, asshown in FIG. 3B, the trapezoidal shape and intensity gradient (shown,for the purpose of illustration, as a gradient in the form of steppedintensities as indicated by the range of colors from black, relativelymore intense, to white, relatively less intense) of trapezoidal shapedregulated light 130 provides a more uniform illumination when theselected area is illuminated at a non-normal angle of incidence.Further, due to the non-normal angle of incidence, in such embodimentsas shown, the use of a trapezoidally shaped regulated light 130 resultsin the illumination of a rectangular shaped area 135.

According to various embodiments, illumination can be provided with arange of intensity variations. For example, embodiments may beconfigured to illuminate the selected area with an intensity variationof 50% or less, including an intensity variation of 10% or less,including an intensity variation of 5% or less, including an intensityvariation of 1% or less, and including an intensity variation at anyvalue between 50% to less than 1%. According to certain embodiments, theselection of an appropriate intensity variation may take into accountany one or more of the following non-limiting factors: the intensityvariation of the light source, the requirements for the targetillumination, and the type of diffractive optical element employed, aswell as other factors such as size, cost, and tolerance limitations ofthe apparatus and/or method. According to certain embodiments, theintensity variation may be a scaled intensity variation.

According to certain embodiments, a range of illumination efficienciesfor illumination of the selected area may be provided. For example,embodiments may be configured to direct at least 1% percent of the lightfrom the light source onto the selected area, including at least 10%percent of the light, including at least 25% percent of the light,including at least 50% percent of the light, including at least 75%percent of the light, including at least 90% percent of the light,including at least 99% of the light and including any percent between 1%and 100%. According to certain embodiments, the selection of anappropriate illumination efficiency may take into account any one ormore of the following non-limiting factors: the number and type ofoptical elements in the apparatus, including the light source and itsintensity, and the intensity requirements for the sample illumination.In certain embodiments, one may factor in the type of power source forthe system, where, for example, relatively high efficiencies may bedesirable for battery powered operation.

In certain embodiments, the present invention may be configured todirect at least 10% percent of the light from the light source onto theselected area and to illuminate the selected area with an intensityvariation of less than 25%. In certain embodiments, the presentinvention may be configured to direct any percent between 10% and 100%percent of the light from the light source onto the selected area and toilluminate the selected area with an intensity variation of any percentless than 25%.

In certain embodiments of the present invention, the apparatus may beconfigured to control a numerical aperture of the regulated lightdirected onto the selected area. In certain embodiments, such a designcan be used to produce a selected depth of field and a selected edgesharpness.

According to various embodiments, virtually any target or selected areamay be illuminated. For example, according to certain embodiments, thetarget may comprise at least one optical fiber bundle, including anoptical fiber bundle comprising separate wells at terminal ends ofoptical fibers of the bundle, and including optical fiber bundles suchas those disclosed in U.S. Pat. No. 6,023,540 to Walt et al., which isincorporated by reference herein in its entirety for any purpose.According to certain embodiments, the target may comprise at least onemicrocard, including, for example, those sold under the trade nameTAQMAN® CYTOKINE GENE EXPRESSION CARDS by Applied Biosystems. Accordingto certain embodiments, the target may comprise at least one of asubstrate, fluidics network, and device, such as those disclosed in U.S.Pat. No. 6,126,899; WO application Ser. No. 97/36681; and U.S. Pat. No.6,272,939, the disclosures of which are incorporated herein by referencein their entirety for any purpose. According to certain embodiments, thetarget may comprise at least one of a glass slide, target array, andmicrowell.

According to certain embodiments, the target may comprise at least oneoptically active species, including at least one optically activespecies chosen from quantum dots and colloidal particles. According tocertain embodiments, one can configure targets to comprise at least oneoptically active species such that one can make measurements of thequantity and/or type and/or change in optical signature of the opticallyactive species in order to measure a baseline and/or result in an assay.

According to certain embodiments, the target may also comprise at leastone luminescent species, including at least one fluorescent species.Luminescent species emit light at a different wavelength than that whichthey absorb, which typically provides easy separation of the excitationlight and the emission light.

According to certain embodiments, the target may comprises at least onerecognition element. Functionally, a recognition element provides and/orhas the ability to discriminate between one or more objects, such as oneor more antigens. The discrimination may be due to any interaction,including, as non-limiting examples, physical, electrical, chemical, andbiological interactions, as well as combinations thereof. In certainembodiments, the at least one recognition element may be chosen fromchemical recognition elements and biochemical recognition elements. Forexample, the target may comprise recognition elements such as thefunctionalized, optically encoded micro spheres of Walt et al.

According to certain embodiments, the selected area of the target mayhave any shape, and may or may not be a single continuous area. Forexample, the selected area may be rectangular, including approximately 1mm×1.5 mm.

According to certain embodiments, the selected area may comprise atleast two or more spatially separate areas. For example, the selectedarea may be at least two wells separated by some distance, and, incertain embodiments of the present invention, the spatially separatewells but not the area between the wells, will be illuminated. Incertain embodiments, the selected area may comprise the wells of amicrotiter plate chosen from microtiter plates having 96, 128, 384, and1536 wells. In certain embodiments, for example, the selected area maycomprise multiple well plates such as those sold by Applied Biosystemsunder the trade names TAQMAN® CYTOKINE GENE EXPRESSION CARDS MICROAMP®384-WELL REACTION PLATES, and MICROAMP® 96-WELL TUBES/TRAY/RETAINERASSEMBLIES. According to certain embodiments, the spatially separateareas may be illuminated simultaneously, sequentially, and/or anycombination thereof. In certain embodiments, rows of spatially separateareas may be illuminated sequentially.

According to certain embodiments, an apparatus is provided that canilluminate a target having microwells. In certain embodiments, theapparatus comprises a target comprising microwells, a light source, afirst lens configured to receive light from the light source, adiffractive optical element configured to receive the light from thefirst lens and to regulate the light into regulated light, and a secondlens configured to receive the regulated light and to direct theregulated light onto a selected area of the target. In certainembodiments, microwells are small assay wells. In certain embodiments,microwells are chosen from 96, 128, 384, 1536 well microtiter plates. Incertain embodiments, microwells are chosen from those described in U.S.Pat. No. 6,023,540; J. A Ferguson et al., Analytical Chemistry, 72, 5618(2000); F. J. Steemers et al., Nature Biotechnology, 18, 91–94 (2000);and D. R. Walt, Science, 287, 451–452 (2000), the disclosures of whichare incorporated herein by reference in their entirely for any purpose.

According to certain embodiments, an apparatus is provided that canilluminate a target having chemically functionalized beads. In certainembodiments, the apparatus comprises a target comprising chemicallyfunctionalized beads, a light source, a first lens configured to receivelight from the light source, a diffractive optical element configured toreceive the light from the first lens and to regulate the light intoregulated light, and a second lens configured to receive the regulatedlight and to direct the regulated light onto a selected area of thetarget. Chemically functionalized beads typically refer to beads, suchas microspheres, that carry one or more functional groups capable ofselective or semiselective chemical interaction with at least onespecies. Non-limiting examples of the functionalities of the one or morefunctional groups include a catalytic functionality, such as anenzymatic functionality; a binding or receptor functionality, such asthat of an antibody; a reactive functionality, such as anoxidation-reduction reactivity; and functionalities that, in thepresence of an analyte, change the optical signature of at least one ofthe bead, species associated with the bead, and species in a sample.

According to certain embodiments, an apparatus is provided that canilluminate a target having at least one self-quenching fluorescenceprobe. In certain embodiments, for example, the target may have anoligonucleotide probe which includes a fluorescent reporter molecule anda quencher molecule capable of quenching the fluorescence of thereporter molecule. In certain embodiments, the oligonucleotide probe canbe constructed such that the probe exists in at least onesingle-stranded conformation when unhybridized where the quenchermolecule is near enough to the reporter molecule to quench thefluorescence of the reporter molecule. In certain embodiments, theoligonucleotide probe can exist in at least one conformation whenhybridized to a target polynucleotide where the quencher molecule is notpositioned close enough to the reporter molecule to quench thefluorescence of the reporter molecule. By adopting these hybridized andunhybridized conformations, the reporter molecule and quencher moleculeon the probe can, for example, exhibit different fluorescence signalintensities when the probe is hybridized and unhybridized. As a result,in certain embodiments it can be possible to determine whether the probeis hybridized or unhybridized based on a change in the fluorescenceintensity of the reporter molecule, the quencher molecule, or acombination thereof. In addition, in certain embodiments because theprobe can be designed such that the quencher molecule quenches thereporter molecule when the probe is not hybridized, the probe can bedesigned such that the reporter molecule exhibits limited fluorescenceuntil the probe is either hybridized or digested. See, for example, U.S.Pat. Nos. 5,723,591; 5,801,155, and 6,084,102, the disclosures of whichare incorporated herein by reference.

According to certain embodiments, the probes include fluorescentmolecules attached to fluorescence quenching molecules by a shortoligonucleotide. In certain embodiments, the probes with the fluorescentmolecules bind to the target molecule, but are broken by the 5′ nucleaseactivity of the DNA polymerase when they are replaced by the newlypolymerized strand during PCR, or some other strand displacementprotocol. When the oligonucleotide portion is broken, the fluorescentmolecule is no longer quenched by the quenching molecule, and emits afluorescent signal. An example of such a system has been described inU.S. Pat. No. 5,538,848, which is incorporated herein by reference, andis exemplified by the TaqMan™ molecule, which is part of the TaqMan™assay system (available from Applied Biosystems).

According to certain embodiments, the probes may be “molecular beacons,”which comprise a fluorescent molecule attached to afluorescence-quenching molecules by an oligonucleotide. When bound to apolynucleotide as double stranded nucleic acid, the quenching moleculeis spaced apart from the fluorescent molecule, and the fluorescentindicator may give a fluorescent signal. When the molecular beacon issingle stranded, the oligonucleotide portion can bend flexibly, and thefluorescence-quenching molecule can quench the fluorescent molecule,reducing the amount of fluorescent signal. Such systems are described inU.S. Pat. No. 5,723,591, which is incorporated herein by reference.

According to certain embodiments, an apparatus is provided that canilluminate a target configured for at least one of hybridization andelectrophoresis. Hybridization is the pairing of complementary nucleicacid strands to make double stranded molecules. Exemplary hybridizationis described in Sambrook et al., eds., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, Chapter 5, Cold Spring Harbor Laboratory Press(1989); Grossman and Colburn, Capillary Electrophoresis Theory andPractice, Chapter 1, Academic Press (1992); Annu. Rev. Biomed. Eng,3:195–223 (2001); Proc. IEEE, 88(12) 1949–1971 (2000); and Annu. Rev.Genomics Hum. Genet. 1:329–60 (2000); Alphey, DNA Sequencing, Chapter 7,BIOS Scientific Publishers Limited (1997), the disclosures of which areincorporated herein by reference in their entirety for any purpose.Electrophoresis is a process in which electrically charged particlesthat are suspended in a solution move through the solution under theinfluence of an applied electric field. Exemplary electrophoresis isdescribed in Grossman and Colburn, Capillary Electrophoresis Theory andPractice, Chapter 1, Academic Press (1992); Annu. Rev. Biomed. Eng,3:195–223 (2001); Chem. Rev. 99, 3081–3131 (1999); Proc. IEEE, 88(12)1949–1971 (2000); J. Biochem. Biophys. Methods 41, 103–119 (1999);Novotny, Capillary Electrophoresis, Biotechnology, 7:29–34 (1996); andU.S. Pat. Nos. 5,741,411, 6,236,945, 5,790,727, and 4,833,827, thedisclosures of which are incorporated herein by reference in theirentirety for any purpose.

According to certain embodiments, an apparatus is provided that canilluminate a target having a target array. In certain embodiments, theapparatus comprises a target comprising a target array, a light source,a first lens configured to receive light from the light source, adiffractive optical element configured to receive the light from thefirst lens and to regulate the light into regulated light, and a secondlens configured to receive the regulated light and to direct theregulated light onto a selected area of the target. A target array isunderstood to be a target composed of more than one sub targets.Non-limiting examples of target arrays include the multiple fiber wellsdisclosed in Walt et al.; U.S. Pat. No. 6,023,540, J. A Ferguson et al.,Analytical Chemistry, 72, 5618 (2000); F. J. Steemers et al., NatureBiotechnology, 18, 91–94 (2000); and D. R. Walt, Science, 287, 451–452(2000), the disclosures of which are incorporated herein by reference intheir entirely for any purpose.

According to certain embodiments an apparatus is provided that isconfigured to perform an assay on a sample. In certain embodiments, theapparatus comprises a target configured to receive the sample, a lightsource, a first lens configured to receive light from the light source,a diffractive optical element configured to receive the light from thefirst lens, and to regulate the light into regulated light, and a secondlens configured to receive the regulated light and to direct theregulated light onto a selected area of the target. According to variousembodiments, the sample may be in any form, such as solid, liquid, gas,and mixtures thereof. Non-limiting examples of such samples includeblood and samples derived from blood, samples of proteins, samples ofnucleic acids, air samples, and/or solutions comprising antibodiesand/or antigens. According to certain embodiments, at least one of thetarget and the sample comprises at least one optically active species.According to certain embodiments, at least one of the target and thesample comprises at least one fluorescent species.

According to certain embodiments, the sample may comprises a “biologicalsample,” which is used in a broad sense and is intended to include avariety of biological sources that contain nucleic acids. Such sourcesinclude, without limitation, whole tissues, including biopsy materialsand aspirates; in vitro cultured cells, including primary and secondarycells, transformed cell lines, and tissue and cellular explants; wholeblood, red blood cells, white blood cells, and lymph; and body fluidssuch as urine, sputum, semen, secretions, eye washes and aspirates, lungwashes and aspirates. According to certain embodiments, samplescomprising fungal and/or plant tissues, such as leaves, roots, stems,and caps, are also within the scope of the present invention. Accordingto certain embodiments, samples comprising microorganisms and/or virusesthat may be present on or in a biological sample are within the scope ofthe invention.

According to certain embodiments, an apparatus is provided that isconfigured to perform an assay on a sample. According to certainembodiments, as shown in FIG. 4, the apparatus comprises a target 70configured to receive the sample. According to certain embodiments, thetarget may comprise a sample holder and/or a sample. The apparatusfurther comprises a light source 10, a first lens 30 configured toreceive light 20 from the light source 10, and a diffractive opticalelement 40. The diffractive optical element 40 is configured to receivethe light 20 from the first lens 10, to regulate the light 20 intoregulated light 50, and to direct the regulated light onto the selectedarea 75 of the target 70. As illustrated by the gradated shading ofregulated light 50, the regulated light may have an intensity gradientto provide more uniform illumination intensity (as compared with lighthaving no intensity gradient) to the selected area 75 when illuminatedat a non-normal angle of incidence, β. In certain embodiments, theregulated light 50 may be shaped with a shape that matches the selectedarea after illumination of the selected area at a non-normal angle ofincidence. According to various embodiments, a second lens, configuredto direct the regulated light onto the selected area, is optional.

According to certain embodiments, as shown in FIG. 4, the selected area75 may be a well, such a well configured to receive a sample. Accordingto certain embodiments, at least one of the target and the samplecomprises at least one optically active species 90. According to certainembodiments, the optically active species 90 comprises at least onefluorescent species.

In certain embodiments, an apparatus optionally may include an opticaldiffuser configured to remove speckle, such as speckle due to theinterference of coherent light. According to certain embodiments, asshown in FIG. 4, an optical diffuser 80 may be located between the firstlens 30 and the diffractive optical element 40. The optical diffuser,however, may be placed anywhere within the apparatus. According tocertain embodiments, the optical diffuser may be any suitable opticalelement which is useful for removing speckle. In certain embodiments,the optical diffuser may comprise at least one of a rotating opticaldiffuser and a light shaping optical diffuser (LSD), such as, forexample, an LSD comprising surface relief holograms with random,non-periodic structures. In certain embodiments, one may use opticaldiffusers from Physical Optical Corporation, including those with thecatalog numbers LSD-KIT-CN-x-y, where x is a diffuser angle chosen from0.5, 1, 5, and 10° and where y is a diameter chosen from 25 and 50 mm.

According to certain embodiments, the invention provides a method toprovide illumination of a target. According to certain embodiments, themethod comprises generating light from a light source, directing thelight with a first lens to a diffractive optical element, generatingregulated light with the diffractive optical element, and focusing theregulated light with a second lens onto a selected area of the target.

According to certain embodiments, the invention provides a method toproduce an optical response. According to certain embodiments, themethod comprises generating light from a light source, directing thelight with a first lens to a diffractive optical element, generatingregulated light with the diffractive optical element, and focusing theregulated light with a second lens onto a selected area of a target,wherein the selected area comprises at least one optically activespecies. According to certain embodiments, the at least one opticallyactive species comprises at least one fluorescent species.

According to certain embodiments, the invention provides an analysismethod. The method comprises generating light from a light source,directing the light with a first lens to a diffractive optical element,generating regulated light with the diffractive optical element,delivering the regulated light onto a selected area of a targetcomprising at least one optically active species, and detecting changesin an optical signature of the at least one optically active species.According to certain embodiments, the at least one optically activespecies may optionally comprise at least one fluorescent species.

It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. An apparatus for illuminating a target, comprising: a light sourcecomprising a coherent light source; a first lens configured to receivelight from the light source; a diffractive diffuser configured toreceive the light from the first lens and to regulate the light intoregulated light; and a second lens configured to receive the regulatedlight and to direct the regulated light onto a selected area of thetarget.
 2. The apparatus of claim 1, wherein the light source is chosenfrom at least one of a laser, an electroluminescent light source, anchemoluminescent light source, an electrochemoluminescent light source,an incandescent light source, a fluorescent light source, an arc lamp,and a light emitting diode.
 3. The apparatus of claim 1, wherein thelight source is chosen from a gas laser, a solid state laser, a fiberoptical laser, and an organic based laser.
 4. The apparatus of claim 1,wherein the light source comprises at least a first light source and asecond light source.
 5. The apparatus of claim 4, wherein said first andsecond light sources respectively emit light having first and secondoptical spectrum, said second optical spectrum being different from saidfirst optical spectrum.
 6. The apparatus of claim 1, where the firstlens is configured to collimate the light from the light source.
 7. Theapparatus of claim 1, wherein the second lens is configured to focus theregulated light to match a size of the selected area.
 8. The apparatusof claim 1, wherein the second lens comprises a lens system configuredto collimate the regulated light and to reduce the regulated light to asize matched to a size of the selected area.
 9. The apparatus of claim1, wherein the second lens comprises an objective lens.
 10. Theapparatus of claim 9, wherein the objective lens is further configuredto collect light from the selected area.
 11. The apparatus of claim 1,wherein the first and second lenses are independently chosen fromrefractive optical elements, reflective optical elements, anddiffractive optical elements.
 12. The apparatus of claim 1, wherein atleast one of the first lens and the second lens is chosen from at leastone cylindrical lens.
 13. The apparatus of claim 1, wherein thediffractive diffuser comprises at least one diffractive optical elementchosen from a transmission hologram, a reflection hologram, a planehologram, a volume hologram, an absorption hologram, and a phasehologram.
 14. The apparatus of claim 1, wherein the diffractive diffusercomprises at least one diffractive optical element chosen from anoptically etched diffractive optical element, an embossed diffractiveoptical element, a molded diffractive optical element, and a chemicallyetched diffractive optical element.
 15. The apparatus of claim 1,wherein the diffractive diffuser is configured to regulate the light andcompensate for at least one of light intensity distributions and shapesof the light due to at least one of the light source and interaction ofthe light with optical elements of the apparatus.
 16. The apparatus ofclaim 1, wherein the diffractive diffuser is configured topost-compensate for effects of elements optically prior to thediffractive diffuser and pre-compensate for effects of elementsoptically subsequent to the diffractive diffuser.
 17. The apparatus ofclaim 1, further comprising an optical diffuser configured to removespeckle.
 18. The apparatus of claim 17, wherein the optical diffuser islocated between the first lens and the diffractive diffuser.
 19. Theapparatus of claim 17, wherein the optical diffuser is a rotatingdiffuser.
 20. The apparatus of claim 1, wherein the regulated light isshaped to match a size and shape of the selected area.
 21. The apparatusof claim 1, wherein the regulated light matches a size of the selectedtarget area after focusing of the regulated light by the second lens.22. The apparatus of claim 1, wherein the regulated light comprises agradient intensity profile for substantially uniform illumination of theselected area at a non-normal angle of incidence.
 23. The apparatus ofclaim 1, wherein the apparatus is configured to substantially uniformlyilluminate a square-shaped selected area of the target at a non-normalangle of incidence; and wherein the diffractive diffuser generatestrapezoidal shaped regulated light having an optical intensity gradientincreasing toward a shorter parallel side of the trapezoidal shapedregulated light.
 24. The apparatus of claim 1, wherein the apparatus isconfigured to substantially uniformly illuminate the selected area. 25.The apparatus of claim 1, wherein the diffractive diffuser regulates thelight such that the regulated light has an intensity distributionsuitable for substantially uniform illumination of the target.
 26. Theapparatus of claim 1, wherein the apparatus is configured to illuminatethe selected area with an intensity variation of less than 50%.
 27. Theapparatus of claim 1, wherein the apparatus is configured to illuminatethe selected area with an intensity variation of less than 10%.
 28. Theapparatus of claim 1, wherein the apparatus is configured to illuminatethe selected area with an intensity variation of less than 5%.
 29. Theapparatus of claim 1, wherein the apparatus is configured to illuminatethe selected area with an intensity variation of less than 1%.
 30. Theapparatus of claim 1, wherein the apparatus is configured to direct atleast 1% percent of the light from the light source onto the selectedarea.
 31. The apparatus of claim 1, wherein the apparatus is configuredto direct at least 10% percent of the light from the light source ontothe selected area.
 32. The apparatus of claim 1, wherein the apparatusis configured to direct at least 25% percent of the light from the lightsource onto the selected area.
 33. The apparatus of claim 1, wherein theapparatus is configured to direct at least 50% percent of the light fromthe light source onto the selected area.
 34. The apparatus of claim 1,wherein the apparatus is configured to direct at least 75% percent ofthe light from the light source onto the selected area.
 35. Theapparatus of claim 1, wherein the apparatus is configured to direct atleast 90% percent of the light from the light source onto the selectedarea.
 36. The apparatus of claim 1, wherein the apparatus is configuredto direct at least 10% percent of the light from the light source ontothe selected area and to illuminate the selected area with an intensityvariation of less than 25%.
 37. The apparatus of claim 1, wherein thetarget comprises at least one optical fiber bundle.
 38. The apparatus ofclaim 1, wherein the target comprises at least one microcard.
 39. Theapparatus of claim 1, wherein the target comprises at least one glassslide.
 40. The apparatus of claim 1, wherein the target comprises anoptical fiber bundle comprising separate wells at terminal ends ofoptical fibers of the bundle.
 41. The apparatus of claim 1, wherein thetarget comprises at least one optically active species.
 42. Theapparatus of claim 41, wherein the at least one optically active speciesis chosen from quantum dots and colloidal particles.
 43. The apparatusof claim 1, wherein the target comprises at least one luminescentspecies.
 44. The apparatus of claim 1, wherein the target comprises atleast one fluorescent species.
 45. The apparatus of claim 1, wherein thetarget comprises at least one of a chemical recognition element and abiochemical recognition element.
 46. The apparatus of claim 1, whereinthe selected area comprises at least two spatially separate areas. 47.The apparatus of claim 1, wherein the selected area is rectangular. 48.The apparatus of claim 1, wherein the selected area is approximately 1mm×1.5 mm.
 49. An apparatus for illuminating a target, comprising: alight source comprising at least one of a gas laser, a solid statelaser, a fiber optical laser, and an organic based laser; a first lensconfigured to receive light from the light source; a diffractivediffuser configured to receive the light from the first lens and toregulate the light into regulated light; and a second lens configured toreceive the regulated light and to direct the regulated light onto aselected area of the target.
 50. An apparatus for illuminating a target,comprising: a light source comprising at least a first light source anda second light source; a first lens configured to receive light from thelight source; a diffractive diffuser configured to receive the lightfrom the first lens and to regulate the light into regulated light; anda second lens configured to receive the regulated light and to directthe regulated light onto a selected area of the target.
 51. Theapparatus of claim 4, wherein said first and second light sourcesrespectively emit light having first and second optical spectrum, saidsecond optical spectrum being different from said first opticalspectrum.
 52. An apparatus for illuminating a target, comprising: alight source; a first lens configured to receive light from the lightsource; a diffractive diffuser configured to receive the light from thefirst lens, to regulate the light into regulated light, and topost-compensate for effects of elements optically prior to thediffractive diffuser and pre-compensate for effects of elementsoptically subsequent to the diffractive diffuser; and a second lensconfigured to receive the regulated light and to direct the regulatedlight onto a selected area of the target.
 53. An apparatus forilluminating a target, comprising: a light source; a first lensconfigured to receive light from the light source; a diffractivediffuser configured to receive the light from the first lens and toregulate the light into regulated light; a second lens configured toreceive the regulated light and to direct the regulated light onto aselected area of the target; and an optical diffuser configured toremove speckle.
 54. The apparatus of claim 53, wherein the opticaldiffuser is located between the first lens and the diffractive diffuser.55. The apparatus of claim 53, wherein the optical diffuser is arotating diffuser.
 56. An apparatus for illuminating a target,comprising: a light source; a first lens configured to receive lightfrom the light source; a diffractive diffuser configured to receive thelight from the first lens and to regulate the light into regulatedlight; and a second lens configured to receive the regulated light andto direct the regulated light onto a selected area of the target,wherein the regulated light is shaped to match a size and shape of theselected area.
 57. An apparatus for illuminating a target, comprising: alight source; a first lens configured to receive light from the lightsource; a diffractive diffuser configured to receive the light from thefirst lens and to regulate the light into regulated light; and a secondlens configured to receive the regulated light and to direct theregulated light onto a selected area of the target, wherein theregulated light matches a size of the selected target area afterfocusing of the regulated light by the second lens.
 58. An apparatus forilluminating a target, comprising: a light source; a first lensconfigured to receive light from the light source; a diffractivediffuser configured to receive the light from the first lens and toregulate the light into regulated light; and a second lens configured toreceive the regulated light and to direct the regulated light onto aselected area of the target, wherein the regulated light comprises agradient intensity profile for substantially uniform illumination of theselected area at a non-normal angle of incidence.
 59. An apparatus forilluminating a target, comprising: a light source; a first lensconfigured to receive light from the light source; a diffractivediffuser configured to receive the light from the first lens and toregulate the light into regulated light; and a second lens configured toreceive the regulated light and to direct the regulated light onto aselected area of the target, wherein the apparatus is configured tosubstantially uniformly illuminate a square-shaped selected area of thetarget at a non-normal angle of incidence; and wherein the diffractivediffuser generates trapezoidal shaped regulated light having an opticalintensity gradient increasing toward a shorter parallel side of thetrapezoidal shaped regulated light.